The goal of this proposal is to understand how kinetochores are dynamically linked to spindle microtubules (MTs) to achieve accurate chromosome segregation and prevent aneuploidy. A major focus is to further advance our knowledge of kinetochore protein architecture and protein conformational changes responsible for: 1) kinetochore microtubule (kMT) formation (end-on MT plus end attachment); 2) pulling and resistive force production coupled to depolymerization and polymerization of attached MT plus- end; 3) control of the spindle assembly checkpoint; 4) correction of attachment errors, such as when an individual kinetochore becomes attached to the ends of spindle MTs from opposite poles rather than from just one pole (merotelic kinetochores); and 5) coordinated motility of sister kinetochores to achieve metaphase alignment at the spindle equator. A major strength of our program has been, and will continue to be, the development and application of new fluorescence microscopy and digital imaging techniques, such as K-SHREC, for measurements of kinetochore protein architecture and conformational changes at nm-scale accuracy and the use of fluorescent bio-sensors for FRET and fluorescence polarization to measure dynamic changes in protein conformation in living cells. Many of our experiments focus on the highly conserved KMN network of proteins (Knl1, Mis12, Ndc80 and complexes) which are kinetochore outer-domain protein complexes that are required for the dynamic and load-bearing characteristics of kMT attachment, control the checkpoint, participate in force generation and implement kMT attachment error correction by being a major target of Aurora B kinase by an unknown tension-sensitive mechanism. Constitutive centromere associated proteins (CCANs), particularly CENP-T and CENP-C, are kinetochore inner-domain proteins that link the KMN network to centromeric chromatin at sites specified by the modified histone H3, CENP-A. The KMN network in turn recruits outer kinetochore proteins, including checkpoint proteins, and other proteins important for kMT attachment, force production and silencing the checkpoint (e.g., CENP-F, the MT motor proteins CENP-E and cytoplasmic dynein /dynactin, and MT associated proteins SKA, EB1, TOG, Mdia3 and potentially Cdt1). Most of our assays of kinetochore protein architectural dynamics use human and other mammalian tissue cells in culture, where kinetochores attach to ~17 kMTs each, or budding yeast, where kinetochores attach to only one kMT.